Cryogenic freezing of liquids

ABSTRACT

The invention relates to method and apparatus for the hyper-rapid freezing of liquid samples. The samples are converted into droplets or vapor and rapidly driven directly onto the surface of a solid or slushed refrigerant.

This application claims priority from U.S. Provisional Application Ser.No. 60/089,683, filed Jun. 17, 1998, which is hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus for preservation ofspecimens by hyper-rapid freezing. More particularly, the presentinvention relates to methods for hyper-rapid freezing of liquidbiological specimens utilizing solid or slushed refrigerants.

2. Description of the Related Art

Freezing has been defined as the solidification of a liquid and may bedivided into two kinds: crystallization and vitrification.Crystallization involves an orderly arrangement of molecules, whilevitrification is the glass-like solidification of solutions at lowtemperature without ice crystal formation. Vitrification can be achievedby increasing the viscosity of the solution and by high speeds ofcooling and warming. Within certain limits, the higher the speed of thetemperature change, the lower the viscosity required to vitrify.

Dubochet, et al. (J. Microscopy, vol. 124, pt. 3, pp. RP3-RP4, 1981)describes novel methods and apparatus for ultra-rapid vitrification ofwater. Successful vitrification of pure water was achieved by sprayingwater onto a wire screen and dropping the screen into liquid nitrogen.Microscopic examination of sections of the frozen film of water revealedthat the regions near the surface had been vitrified into anoncrystalline glass, although regions where the water film was thickerhad frozen into small crystals. Apparently, heat from the water filmcould be transferred rapidly enough into liquid nitrogen to freeze waterinto a glass structure before ice crystals had a chance to form. The keyto success of the experiment was the high surface to volume ratio (film)of the water, and the rapid decrease in temperature provided by theplunge into liquid nitrogen.

Rapid water vitrification requires extremely rapid freezing rates. Icecrystal size depends primarily on how rapidly heat is removed from thefreezing sample. Faster freezing rates lead to smaller ice crystals. Ifheat energy can be removed quickly enough, then ice crystals will becomevanishingly small, and, at fast enough freezing rates, ice crystals willnot form at all. Instead, the water molecules slow their movement soquickly they do not have time to form a crystal lattice, and willsolidify into a disorganized glass-like structure, i.e. vitrify.

Water molecules are very polarized, and therefore easily form crystallattices when frozen. Compared to other compounds, the rate of freezingpure water into a vitreous state is extraordinarily rapid on the orderof −1,000,000° F./sec. This freezing rate is so high that in the past,vitrification of pure water was thought to be virtually impossible, andcurrently only a few technological methods exist that are capable ofvitrifying very small quantities of water.

Preservation of biological specimens for future use has long been a goalof the scientific community. Successful methods such as freezing andfreeze drying have been achieved for simple biological specimens. Manycomplex macromolecules and whole cells show reduced activity orviability following freezing or freeze drying by conventional methods.

In general, cell injury or cell death from freezing occurs from one (orboth) of two mechanisms. The first mechanism is growing ice crystals,which injure cell membranes and organelles due to their sharp edges.Usually, the ice crystals form and grow primarily in the extracellularspace and encroach upon the cell from outside. As freezing progresses,the (relatively) large ice crystals damage or puncture the cell membraneand distort the shape and overall structure of the entire cell. As thefreezing process nears completion, intracellular ice formation may alsoform, with internal ice crystals causing physical damage to organellesand other cellular structures.

The second mechanism is internal cell “poisoning” which results fromvery high concentrations of intracellular solutes, created by osmoticdehydration. As freezing progresses, extracellular ice crystals form andgrow larger. The ice is relatively pure water, and ice crystal growthconsumes extracellular liquid water which then increases theextracellular solute concentrations. An osmotic force is created betweenthe intracellular and extracellular spaces which drives water across thecell membrane out of the cell. As extracellular water is consumed by icegrowth, it is replaced by intracellular water and the cell becomesosmotically dehydrated. Increasing concentrations of various solutesinside the cell eventually reach toxic levels, damaging or killing thecell.

Both mechanisms operate simultaneously during cell or tissue freezingprocesses using most current technologies. Balancing the influence ofeach mechanism to minimize cell damage during freezing and thawing isthe goal of any cryopreservation process.

High cooling rates are necessary to freeze biological specimens whileavoiding ice crystal formation and maintaining in situ diffusiblechemical components. The conventional method is plunging cryoprotectedbiological specimens into a cooling liquid such as liquid nitrogen. Thismethod may be used in the absence of cryoprotectants if the specimensare thin and mounted on low-mass sample holders and the coolant aroundthe sample is renewed fast enough to prevent the formation of a gaseouslayer which would limit heat transfer (Escaig, J. Microscopy, vol. 126,pt 3, pp. 221-229, 1982). Another method in which cryoprotectants arenot required is contacting the specimen with a metal block cooled to thetemperature of liquid nitrogen or helium (Escaig 1982; Sitte, et al., J.Microscopy, vol. 111, pt 1, pp 35-38, 1977). Problems associated withthese methods include the difficulty of making suitable specimen holdersand the lack of reproducibility of freezing.

Another approach to preserving biological specimens is freezing underhigh pressure. Conaway (U.S. Pat. No. 4,688,387) teaches placing thebiological specimen into a pressure vessel, applying high pressure tothe specimen, and then placing the pressure vessel into liquid nitrogento achieve cooling. A disadvantage of this method is that long termstorage at high pressure and low temperature is not convenient.Therefore, once the specimen is frozen, the pressure is released and thespecimen is stored at low temperature. However, the specimen must beplaced under high pressure again prior to thawing.

Most protein and peptide specimens exhibit poor survival during theconventional freeze-thaw or freeze-dry processes, with low recoveries of40% to 90% of the original specimen. This is attributed to physicalinjury and loss of structural integrity of the protein molecule,presumably from water crystal or osmotic damage during the freezingprocess.

Cryopreservation agents may be added or controlled freezing rates usedin order to maximize cell survival. Cryopreservation agents, such asDMSO or propylene glycol, reduce the freezing temperature point and icecrystal size, and at optimal concentrations the cryopreservation agentswill significantly increase cellular freeze-thaw survival. However,excessive concentrations of cryopreservatives are toxic to cells andtissue, limiting their usefulness. Controlled freezing rates allowbetter optimization of the two cell damage mechanisms at different timesduring the freezing process, and use of plateau and stopping points topermit ice crystal seeding further enhances cell survival for someprocesses. Optimal freeze-thaw survival for some current applicationsrequires a complex process of cell preparation, changing concentrationsof cryopreservation agents, multi-step freezing rates, multiple icecrystal seedings, and finally a rapid plunge into liquid nitrogen orother cryogenic liquefied gas to vitrify the remaining liquid that hadnot already frozen into ice crystals. Complex thawing protocols may alsobe required to minimize ice crystal or hyper-concentrated solutes fromfurther damaging the cells.

Several types of biological specimens have been successfully frozen andthawed using cryopreservatives and various freezing rates. Examplesinclude plant material, tissue culture cells, sperm and embryos.Oocytes, however, are particularly difficult to cryopreserve for thefollowing reasons:

1) High volume-to-surface ratio. Oocytes are the largest cells of themammalian body. During equilibration, high concentration gradients ofcryoprotectants may arise resulting in toxic damage in one part of thecell and insufficient protection in the other part of the cell. Osmoticeffects are also increased and during equilibration and dilution oocytesmay suffer extreme changes of shape resulting in possible damages ofmembranes and cytoskeleton.

2) Chilling injury. In bovine oocytes, low temperature sensitivity isattributed to two different factors. Besides damage of the numerouslipid droplets, the meiotic spindle of the metaphase II oocytes suffersserious injury when cooled to 4 or even 25° C. Unlike the lipidalteration, damage to the spindle after short-time low-temperatureexposure is reversible, but may even so disturb the subsequentfertilization process.

3) Alteration of the zona pellucida. Cooling may result in decreasedfertilization rates caused by premature cortical granule exocytosis andzona hardening.

4) Cumulus cell removal. Both immature and mature oocytes form afunctional unit with the attached cumulus cell layers. Unlike the invivo process, the cumulus investment is beneficial for in vitrofertilization either by promoting sperm capacitation or preventingpolyspermy. However, these layers and the accumulated glycoproteinsreduce the speed of cryoprotectant penetration either at equilibrationor at dilution. In a group of oocytes, partial removal of cumulus cellsis very difficult to perform uniformly, and the differences may resultin uneven cryoprotectant penetration (Vajta, IETS Newsletter, June 1997,http://www.iets.uiuc.edu/news/iets697.html#featured). The damagesustained is often enough to kill the cell or prevent if fromsuccessfully fertilizing.

Martino, et al (Biology of Reproduction, vol. 54, pp 1059-1069, 1996)discloses a method for cryopreserving bovine oocytes in which oocyteswere placed in a cryopreservative medium, and placed either in straws oron electron microscope grids. The straws were plunged into liquidnitrogen and the grids were either plunged into liquid nitrogen ornitrogen slush. Martino, et al. report that cooling rates achieved withgrids were much higher than with straws. To test whether the use of evenfaster cooling rates would improve survival, nitrogen slush was comparedto liquid nitrogen for freezing oocytes on grids. Survival rates basedon morphology, cleavage and blastocyst formation were higher for oocytesfrozen in liquid nitrogen compared to those frozen in nitrogen slush.

Successful cryopreservation of biological specimens without the risk ofcryoprotectant toxicity and loss of viability common with currentmethods would have significant clinical and scientific applications.Successful cryopreservation of human oocytes would have significantclinical applications, including frozen quarantine of donor eggs toprevent HIV or hepatitis transmission to the recipient, long-termstorage of oocytes for chemotherapy patients who wanted to later attemptpregnancy, or for women undergoing oophorectomy for benign disease, orfor short-term storage for women with ovarian hyper-stimulationsyndrome.

The practice of freezing and storing sperm and embryos has achieved acertain amount of success. Freezing and thawing unfertilized oocytes,however, has not generally been successful for reasons set forth above.The ability to freeze and store oocytes and sperm separately would avoidthe legal complications regarding custody and maintenance of frozenembryos. The instant invention is particularly useful for freezingunfertilized oocytes.

SUMMARY OF THE INVENTION

An embodiment of the instant invention is a method of rapidly freezingand storing water or liquid samples without the use of cryoprotectantsto minimize or eliminate damage from ice crystals or osmotic forces,allowing very high recovery rates upon thawing. The liquid sample istransformed into very small drops which are contacted directly with arefrigerant which is at least partially solidified. The refrigerant maybe a solid undergoing a solid-to-liquid phase change, or a solid-to-gasphase change. This method is especially useful for substances that aresusceptible to ice crystal or osmotic damage, such as proteins,peptides, and other macromolecules.

A further embodiment of the invention is a method of stopping a chemicalor biological reaction by rapidly freezing the reaction mixture throughdirect contact with a refrigerant which is at least partiallysolidified. Molecular slowing occurs almost instantly, preserving thecompounds in mid-reaction. The various molecular steps of the reactionare frozen in place, and can be analyzed by studying the vitrifiedsample.

A further embodiment of the invention is to provide a method forlyophilizing chemical and biological compounds or compositions whileretaining structural integrity. Reconstitution of the substance willrestore the original liquid sample with little or no damage. Specificapplications of this type of lyophilization include freeze driedfoodstuffs (coffee, milk, juices, etc.), medical supplies (vaccines,antibiotics, etc.) and laboratory processes (protein extractions,molecular biology samples, etc.).

An additional embodiment of the invention is a method for production offine-grained or amorphous solids by direct deposition of a vaporsubstance onto a cryogenic gas which is at least partially solidified.

Another embodiment of the invention is an apparatus for rapid freezing aliquid sample. The apparatus comprises a means for driving the liquidsample directly onto a refrigerant which is at least partiallysolidified. Another embodiment of the apparatus is one which furthercomprises a means for transforming the liquid sample into very smalldrops prior to driving them onto the refrigerant.

A method was developed for rapidly freezing a sample in which primaryand secondary refrigerants in thermal contact are used to achieve thestate of at least partial solidification of the primary refrigerant.Another method for achieving a state of at least partial solidificationof the primary refrigerant is evaporative cooling by placing therefrigerant in a vacuum chamber and subjecting it to low pressure.

Various additional features can be included in further embodiments ofthe invention. One such feature is an apparatus with multiple freezingchambers. Another feature is an apparatus with a continuously renewedsolidified primary refrigerant surface. Additional features includeapparatus for direct injection of sample onto solid or into partiallysolidified refrigerant.

An additional embodiment of the invention is a method for rapidlyfreezing non-liquid samples by contacting them directly with arefrigerant which is at least partially solidified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are illustrations of the basic method of this invention.

FIG. 2 is a cross-sectional view of an embodiment of the hyper-rapidfreezing device involving primary and secondary refrigerants.

FIG. 3 is a perspective view of a device for hyper-rapid freezing with acontinuously renewed solidified argon surface.

FIG. 4 is a perspective view of a device for continuous hyper-rapidfreezing.

FIG. 5 is a perspective view of a device for hyper-rapid freezing with acontinuously renewed solidified argon surface.

FIG. 6 is a cross-sectional view of a device for hyper-rapid freezingwith a continuously renewed solidified argon surface.

FIG. 7 is a perspective view of a device for hyper-rapid freezingutilizing mechanical refrigeration.

FIGS. 8, 9 and 10 are perspective views of devices for hyper-rapidfreezing utilizing centrifugation.

FIG. 11 is a cross-sectional view of a device for hyper-rapid freezingwith multiple sample freezing chambers.

FIG. 12 is a perspective view of a device for hyper-rapid freezingutilizing direct injection of sample onto solid refrigerant.

FIG. 13 is a cross-sectional view of a device for hyper-rapid freezingutilizing direct injection of sample into partially solidifiedrefrigerant.

FIG. 14 is a perspective view of a vacuum chamber for evaporativecooling of refrigerant.

DETAILED DESCRIPTION OF THE INVENTION

The most basic element of this rapid freezing method is bringing smallliquid drops or particles into direct contact with a refrigerant whichis at least partially solidified, and collecting the frozen liquid. Avariety of devices and processes can be used to implement this basicmethod. The simplest version of this embodiment is spraying a nebulizedor atomized liquid directly onto the refrigerant's surface. Thisembodiment of the invention is illustrated in FIGS. 1A and 1B. Anebulizer 10 converts the sample into very small droplets 11 whichcontact solid 12 or slushed 13 primary refrigerant.

As used herein, the terms “collect” and “collecting” shall refer to theprocess of recovering the frozen material for transport. Thus, the term“collecting” will include creating the frozen material in a containersuitable for transport and storage, such that the freezing and thecollecting steps occur concurrently.

The phase change of the refrigerant from solid to at least partiallysolidified to liquid is a key element of this process. The phrase “atleast partially solidified” is intended to encompass completely solidand slush phases. The term “slush” is intended to encompass liquid thathas just begun to solidify through equal parts solid and liquid up tothe point just before complete solidification. In a slush, the ratio ofsolid to liquid components is such that movement of particles of solidis controlled by the liquid component. The solid and liquid componentsof a slush may be the same element or compound or may be differentelements or compounds. A slush includes stable mixtures of liquid andsolid substantially at equilibrium. When the solid and liquid componentsare different elements or compounds, the elements or compounds must becompatible in terms of providing an environment suitable for freezingthe sample.

A large number of substances can be employed as the refrigerant,depending on the temperature range desired and the composition of theliquid drops to be frozen. It should be understood that “refrigerant”includes substances with a temperature less than or equal to 0° C. Whentwo or more refrigerants are used, the entire system has a temperatureless than or equal to 0° C. Refrigerants can be selected from severalbasic types, including chemical elements and compounds, organicsubstances or mixtures of these items. Examples of elements includehelium (under pressure), hydrogen, nitrogen, argon, neon, krypton,xenon, oxygen, mercury, gallium, and lead. An example of a compound iswater and carbon dioxide (under pressure). Examples of organicsubstances include propane, benzene, ethanol, methanol, and Freon.Mixtures of two or more elements and/or compounds mixed together toalter melting temperature or other physical characteristics can also beused as the refrigerant; e.g. Water+NaCl→Brine,Oxygen+Nitrogen→Slushed/Solid Air, Ethane+Propane.

Any substance or compound can be used as the refrigerant for thisfreezing method as long as it can rapidly absorb heat from liquiddroplets by its phase change from solid to partially solidified toliquid. The melting temperature of the refrigerant must be lower thanthe liquid droplet freezing temperature, and the heat of fusion absorbedby the refrigerant (along with some additional heat absorbed by theliquefied refrigerant in some cases) must be greater than the heat offusion released by the freezing liquid droplets.

A variation of this freezing method involves the use of sublimatingcompounds as the refrigerant. The phase change from solid to liquid isthe basic process for absorption of heat from freezing droplets, but thephase change from solid to gas of the refrigerant can also be used. Anexample is spraying nebulized liquid droplets directly onto or into thesurface of solid carbon dioxide. A disadvantage of this method is thegeneration of an insulating gas layer around the droplets, which mayslow the rate of freezing. This disadvantage can be overcome usingdevices that rapidly drive the liquid droplets into the solidrefrigerant in order to maintain direct contact between their surfaces.

The production of solid or slushed refrigerants, especially in thecryogenic temperature range, requires mechanical means or a secondaryrefrigerant. The mechanical means may be in the form of a refrigerationmeans or a vacuum chamber. Liquid refrigerant is placed in a vacuumchamber and placed under partial vacuum to achieve at least partialsolidification of the refrigerant. The secondary refrigerant can be anyheat-absorbing substance held at a temperature lower than the freezingpoint of the primary refrigerant. A secondary refrigerant is used tofreeze the primary refrigerant into a solid or slushed state either bydirect contact or by indirect contact through a container. Freezing ofthe primary refrigerant may be a one-time single event, or can occur asa repetitive or continuous renewal process. Selection of appropriateprimary and secondary refrigerants results in production of an optimalslushed primary refrigerant or may be used to change the workingtemperature of the primary solid refrigerant to maximize liquid dropletfreezing rates. For example, frozen argon held at the temperature ofliquid helium will absorb much more heat more rapidly from liquiddroplets than frozen argon held at liquid nitrogen temperature. Becauseof the wide range of freezing temperatures for various elemental,compound and organic refrigerants, numerous combinations of primary andsecondary refrigerants are possible. The secondary refrigerant for onerapid-freezing application can be used as a primary refrigerant foranother application. Examples of primary and secondary refrigerants areprovided in Table 1.

TABLE 1 PRIMARY REFRIGERANT SECONDARY Melting Melting REFRIG- PointPoint Hydro- Oxy- Nitro- Pro- Butene- Eth- Kryp- Etha- Mer- Gal- ERANT °C. ° C. gen Neon gen gen pane Argon 1 ane ton nol Xenon cury Water liumHelium −272.20 −268.90 ◯ ◯ X X X X X X X X X X X X Hydrogen −259.14−252.87 X X X X X X X X X X X X X Neon −248.67 −246.05 X X X X X X X X XX X X Oxygen −218.40 −182.96 ◯ ◯ ◯ ◯ ◯ X X X X X X Nitrogen −209.86−195.80 X X X X X X X X X X Propane −189.69 −42.07 ◯ ◯ ◯ ◯ ◯ X X X Argon−189.20 −185.70 — — X X X X X Butene-1 −185.35 −6.30 — — — — ◯ X XEthane −183.30 −88.63 ◯ ◯ ◯ X X X Krypton −156.60 −152.30 X X X X XEthanol −117.30 78.5 — — — — Xenon −111.90 −107.10 X X X Mercury −38.87356.58 X X Water 0.00 100.00 ◯ Gallium 29.78 2403.0 X = primaryrefrigerant freezes in secondary refrigerant ◯ = primary refrigerantfreezes in chilled secondary retrigerant — = primary refrigerant barelyfreezes in chilled secondary refergerant

Referring to FIG. 2, the hyper-rapid freezing apparatus is generallyreferred to at 14. It comprises a nebulizer 15 positioned inside anebulizer column 16 which fits into an opening 21 in a hollow sphere 17containing argon. The sphere is placed into a container 19 of liquidnitrogen. The liquid nitrogen causes the argon inside the sphere tofreeze along the walls. A sample is delivered to the nebulizer 15 viathe nebulizer column 16 and sprayed onto the solid argon on the innerwalls of the sphere 17. The frozen sample accumulates in the liquidargon at the bottom of the sphere 17 and is collected by removing thenebulizer column 16 from the sphere 17 and pouring out the liquid argon.Alternatively, the sphere 17 can be used for storing the frozen sampleby removing the nebulizer column 16 and plugging the opening 21.

An embodiment of the invention is illustrated in FIG. 3. The hyper-rapidfreezing apparatus 22 comprises a rotating drum 23 filled with liquidsecondary refrigerant. The drum 23 is positioned inside a housing 25half-filled with liquid primary refrigerant such that the drum 23 ispartially submerged in the primary refrigerant. The apparatus furthercomprises a series of nebulizers 27-29 positioned over the exposedsurface of the drum 23. As the drum 23 rotates, the secondaryrefrigerant inside causes a layer of frozen primary refrigerant to formon the exposed surface. Sample is sprayed from nebulizers 27-29 onto thefrozen primary refrigerant on the surface of the drum 23. As the drum 23rotates, the frozen sample floats off into the liquid primaryrefrigerant. The frozen sample is collected by skimming, sedimentation,or filtration of the liquid primary refrigerant, depending on thedensity of the frozen sample.

Another embodiment of the invention is illustrated in FIG. 4. Housing 30has a sloped upper surface comprising grooves 31. The end walls of thegrooves have openings 32 connected to a pipe 33 at the upper end, and toa trough 34 at the lower end. The housing 30 is hollow with inlet 35 andoutlet 36 ports for circulating liquid secondary refrigerant through thehousing 30. The device further comprises multiple nebulizers 37, onepositioned over each groove. Liquid primary refrigerant is transportedfrom the pipe 33 through the openings 32, down the grooves 31 where itfreezes due to the liquid secondary refrigerant circulating through thehousing 30. Sample is sprayed onto the frozen primary refrigerant in thegrooves, causing the refrigerant to melt and flow into the trough 34.

A further embodiment of the invention is illustrated in FIG. 5. Housing38 is divided into two sections, one filled with liquid secondaryrefrigerant, and the other partially filled with liquid primaryrefrigerant. A rotating metal disk 39 is positioned half in one sectionand half in the other section such that the disk is in contact with boththe primary and secondary refrigerants. The secondary refrigerant coolsthe metal disk causing a layer of frozen primary refrigerant to form onthe exposed surface of the disk. The device further comprises one ormore nebulizers 40 positioned in the housing section containing primaryrefrigerant such that sample is sprayed onto the frozen primaryrefrigerant coating the exposed portion of the disk.

Another embodiment of the invention is illustrated in FIG. 6. The devicecomprises a vertical cylindrical pipe 41 within a larger cylindricalcontainer 42. The container 42 comprises a lower section filled withliquid secondary refrigerant and an upper section. The pipe ispositioned such that the top opening 43 is in the upper section of thecylindrical container 42 and the bottom opening 44 is below the lowersection of the cylindrical container. Insulation 45 surrounds thecylindrical container. A conduit 46 connected to the lower section ofthe cylindrical container 42 transports the liquid secondaryrefrigerant. A nebulizer 47 is positioned over the top opening 43 in thepipe 41. Liquid primary refrigerant is forced under pressure up into thepipe. The primary refrigerant freezes as it passes through the lowersection of the cylindrical container, and is extruded out of the topopening of the pipe. Sample is sprayed onto the extruded primaryrefrigerant.

Primary refrigerants in slushed or solid forms can be produced (frozen)or held at working temperatures by mechanical refrigeration devices.Although generally more expensive than the use of secondaryrefrigerants, the mechanical devices allow greater control of theworking temperature of the primary refrigerant. An embodiment of theinvention utilizing mechanical refrigeration is illustrated in FIG. 7.The device comprises a source of mechanical refrigeration 48 connectedto one or more containers of primary refrigerant 49. Depending on thetemperature achieved by the mechanical refrigeration source, therefrigerant will be either in the slushed or solid state. A nebulizer 50is positioned above the container of refrigerant.

The physical size of the sample liquid droplets to be frozen has asignificant influence on the freezing rate achieved by this method. Ingeneral, the smaller the liquid droplet, the faster it will freeze.Nebulizer type and control can be used to obtain optimal drop or dropletsizes for individual applications of the freezing method.

A large number of nebulizing, atomizing and vaporizing devices usingdifferent operating principles are available, any of which can be usedto produce liquid droplets in a variety of sizes and rates for thisfreezing process. These include spray nozzles, pressurized smallapertures, gas driven nozzles, fine screen methods, ultrasonic devices,and drip nozzles. Selection of an appropriate device depends on thespecific freezing application and desired drop size, e.g. biological orfragile specimens may be damaged by some nebulizers and may require moregentle types such as glass-fret or fine screen nebulizers.

A flow cytometer can also be used for producing liquid droplets ofspecific sizes. A flow cytometer can be used to achieve individual dropscontaining a single cell, or specific numbers of cells. The amount offluid per cell can also be controlled to achieve optimal freezingconditions which provide for optimal viability upon thawing. The flowcytometer may be communicatively coupled to a container containing asuspension of biological material to be frozen.

The liquid droplet heat of fusion and droplet-to-primary refrigeranttemperature gradient are the factors determining the total amount ofheat to be removed from the droplet to achieve complete freezing. Therate of freezing depends on these factors and the specific heat and thethermoconductivity of the droplet and the primary refrigerant. Thefreezing rate can be altered by lowering the initial temperature of thedroplets, or by changing the heat of fusion by adding solutes. Forexample, most biological specimens in liquid solutions can be precooledto within a few degrees above the freezing point without significantdamage, which would reduce the total amount of heat to be removed fromthe liquid droplets and increase the rate of freezing at the time ofcontact with the primary refrigerant.

Cryopreservatives may be added to the liquid samples to improve thesurvival of biological or other specimens. For freezing rates that donot achieve vitrification of the sample, ice crystal or frozen crystalsizes can be minimized by the addition of a cryopreservative.

The velocity of droplet impact into the primary refrigerant has asignificant influence on the freezing rate of the droplet. A slowapproach and slow impact of the droplet will result in slower freezingrates with more crystal content or larger crystal sizes, or even resultin prefreezing of the droplet before impact with the primary refrigerantas the droplet passes through the temperature gradient above the surfaceof the refrigerant. Increasing the velocity of the droplets beforeimpact will result in more rapid freezing rates, and this can beachieved by increasing the pressure of spray or gas nebulizers, or bydecreasing the distance between the nebulizer and the primaryrefrigerant. Fragile specimens or biological specimens may be damaged bythe high fluid shear forces of high pressure nebulizers, so increasingthe velocity of these nebulized specimen droplets would require lowpressure/velocity nebulization followed by acceleration of the dropletsusing injection of a gas carrier into the nebulizer stream or bycentrifugation of the stream. A high humidity or water saturatedatmosphere, and very fast spray velocities can be used to minimizeevaporation of the sample drop. Rapid spray velocities limit the amountof time for evaporation to occur, and increase the temperature gradientnear the refrigerant surface, reducing the chance of precooling thesample. Because evaporation of a liquid droplet is increased on theforward side when it is sprayed into a still atmosphere, furtherrefinements of the designs involved spraying nebulized droplets into astream of humidified or saturated air or other gas moving in the samedirection at about the same velocity, with the air stream and nebulizedstream directed onto the refrigerant surface.

Direct injection of nebulized droplets into the solid or slushed primaryrefrigerant can maximize the freezing rate for some applications. Thiscan be achieved by inserting the aperture of the pressure or spraynebulizer into the refrigerant slush or onto a solid. As the liquidencounters the refrigerant, it breaks up into small droplets, and thechaotic flow of the droplets through the slush or solid increases thetransfer of heat from the droplets resulting in very high freezingrates. Nebulizer apertures may remain stationary when inserted intoslushed refrigerants, but optimal results from solid refrigerants mayrequire moving the nebulizer aperture along the surface of the frozenrefrigerant.

An embodiment of this variation of the invention is illustrated in FIG.8. The device comprises a centrifuge 51 with paddles 52 holding solidprimary refrigerant 53 connected to the rotor 54. One or more nebulizers55 are positioned in the walls of the centrifuge such that sampledelivered to the nebulizers is sprayed onto the solid refrigerant as therotor turns.

A variation of the embodiment utilizing a centrifuge is illustrated inFIG. 9. A nebulizer 56 is positioned over a centrifuge 57 containingsolid primary refrigerant. Sample is sprayed from the nebulizer onto thespinning surface of refrigerant and the frozen droplets of sample moveto the outer edge, leaving the center of solid refrigerant open toreceive more sample.

Another variation of the embodiment utilizing a centrifuge isillustrated in FIG. 10. A column 58 attached to a rotating head 59comprising multiple horizontal nebulizers 60 is positioned over acylindrical container 61 with solid refrigerant coating the walls.Sample is delivered to the nebulizers through the column and sprayedonto the solid refrigerant coating the walls of the container as thehead rotates.

A further embodiment of the invention is illustrated in FIG. 11. Thedevice comprises a housing 62 filled with liquid secondary refrigerant.One or more hollow rotating cylinders 63 are positioned such that theyare submerged in the liquid secondary refrigerant. Inlet tubes 64 forsample transfer are connected to respective cylinders. Nebulizers 65 arepositioned on the tubes inside the cylinders to deliver sample to theinside walls of the cylinders. Liquid primary refrigerant is placedinside the cylinders. As the cylinders rotate, the primary refrigerantfreezes on the walls. Sample sprayed onto the cylinder wall freezes anddrops into the remaining liquid refrigerant at the bottom of therotating cylinder. When the cylinders are removed from the housing theymay be used for storing the frozen sample.

An embodiment of the invention utilizing direct injection of sample ontosolid refrigerant is illustrated in FIG. 12. The device comprises arotating cylindrical receptacle 66 with solid refrigerant coating theinner walls, and a sample delivery tube 67 with an injection aperture 68at the end. The delivery tube is positioned such that the injectionaperture is in close proximity to the solid refrigerant coating thereceptacle walls.

Another embodiment of the direct injection apparatus is illustrated inFIG. 13. The device comprises a receptacle 69 holding slushedrefrigerant. A cylinder 70 with small injection apertures 71 located atone end is positioned in the receptacle such that the injectionapertures are submerged in the slushed refrigerant. Pressurized sampleis delivered through the injection apertures in the cylinder into theslushed refrigerant. Alternatively, the device may comprise an injectionapparatus 72 connected to the bottom of the receptacle 69. In thisembodiment, the receptacle has small injection apertures 73 in thebottom. Sample is injected into the slushed refrigerant in thereceptacle by means of nebulizer jets 74 in the injection apparatus.

One of the advantages of this rapid freezing method is the very highfreezing rates achieved at atmospheric pressure. However, some specialapplications may be optimized by increasing the pressure at the droplet-refrigerant interface. High pressure can be used to produce a solidifiedor slushed primary refrigerant (i.e. liquid helium) or increase thethermoconductivity of the refrigerant or droplets. Enclosing the basicapparatus (nebulizer and primary refrigerant) in a pressure chamberwould be a typical method to achieve this process. Other specialapplications may require reducing the pressure below atmospheric, and asimilar vacuum chamber would be used in these cases. A vacuum chamberfor producing solid or slushed primary refrigerant is shown in FIG. 14.A container 75 of liquid primary refrigerant is placed inside a vacuumchamber 76 attached to a vacuum pump 77. A nebulizer 78 is positionedover the container 75 of primary refrigerant. The low pressureconditions created by the vacuum pump cause evaporative cooling of theprimary refrigerant.

Most of the individual methods for rapid freezing of nebulized liquidsare limited by the output of the nebulizer. The rate that the liquidsample is frozen is determined by the maximum flow achieved through thenebulizer, or in some instances, the rate of production of delivery ofthe primary refrigerant. The rate of production of frozen particles byany unit (made up of a nebulizer and primary refrigerant delivery) islimited to an inherent maximum. In order to increase the production offrozen particles, the number of freezing units can be increased, andhigh outputs can be achieved by running a large number of identicalfreezing units in parallel. In general, the output of the nebulizer isthe limiting component of each unit, so a typical system would be madeup of several nebulizers sharing a central primary refrigerant delivery(or possibly several parallel refrigerant delivery units).

The process for extremely rapid freezing of liquids has a large numberof industrial, medical, biological, and research applications.Industrial applications of this process include freezing liquidcompounds with minimal chemical damage to the compound, lyophilizationwith high recovery rates, and creation of small crystal or amorphoussolids with special transparency, electrical, thermoconductive, or otherphysical properties. Medical and biological applications includecryopreservation of cells, especially oocytes, and tissue with minimalice crystal or osmotic damage, or production of minimal-damage samplesfor microscopic or electron microscopic examination. Researchapplications include production of vitreous ice, and freeze-stoppingchemical or biological reactions. In order to achieve the extremelyrapid cooling rates required for these applications, heat must beremoved very quickly from the liquid samples. Barriers to rapid heatextraction from the liquids, such as insulating gas envelopes or largethermoconductive distances across the liquid, must be eliminated orminimized. These barriers are overcome or minimized by the freezingprocess of the instant invention. The insulating gas envelope around aliquid sample immersed in a liquid refrigerant is replaced by a highthermoconductive interface with a solid or slushed refrigerant. Heatextracted from the liquid sample is used to melt the refrigerant into aliquid instead of boiling it into an insulating gas layer. The heatextraction barrier resulting from large thermoconductive distancesacross the liquid sample is minimized simply by reducing the liquidsample to small drops, droplets, or a nebulized mist. Reducing theliquid sample to droplets or mist also dramatically increases thesurface area (i.e. the heat flux interface available) of the liquid,further increasing the rate of cooling.

The simplest version of this process involves nebulizing a liquidsample, then rapidly driving the nebulized stream onto or into a solidor slushed refrigerant. The nebulized liquid sample is very rapidlyfrozen into tiny solid particles which can then be easily collected andstored.

Beneficial features of this freezing process include use of therefrigerant's solid-to-liquid phase change, very rapid freezing at alarge scale, ease of sample separation and storage, low-cost materials,simplicity, and variable freezing rates, along with inert nonreactivecomponents for most applications. These features solve or improve on thelimitations imposed by current prior-art freezing methods.

The solid-to-liquid phase change of the refrigerant bathes the sample ina liquid for faster heat transfer (higher thermoconductivity) instead ofa boiled gas envelope with lower thermoconductivity which occurs whenliquid refrigerants are used. This concept is general, encompassing anycryogenic frozen or slushed gas as the refrigerant. Frozen gases can beheld at any temperature below the freezing point, and slushed gases canbe held at the fusion temperature corresponding to any ambient orapplied pressure. Samples to be frozen are typically liquid at anyconcentration, but may also be non-liquid as long as they have thecapacity to be frozen by the solid or slushed refrigerant selected.Non-liquid samples include metals, oil-based organic compounds, lipids,plastics, and silicone substances, or others not limited to thesecategories. Samples can be immersed or applied to the refrigerant in theform of thin films, in vials, tubes, or other containers, or as drops,sprays, droplets, or in nebulized or atomized forms. Optimal hyper-rapidfreezing results are obtained with the optimal combination ofrefrigerant type (gas element or compound), refrigerant phase (solid orslushed), refrigerant temperature, sample type (liquid, non-liquid),sample phase (liquid, nebulized, atomized, vapor), sample temperature,and the method of application (direct spray, drop, air stream).Vitrification of water or other liquids directly from the vapor phase isachieved by blowing a gas saturated with the liquid vapor directly ontoa solidified gas surface (example—air saturated by water vapor).

A method was devised to produce a liquid-solid refrigerant slush. Themelting point of nitrogen is −209.86° C., which is the slush temperatureat one atmosphere pressure. A refrigeration method or colder substancewould be needed to reduce the commercially available liquid nitrogen (atabout −195.8° C.) temperature to the freezing point.

Review of the physical properties of gases revealed that argon, krypton,and xenon have freezing points above that of liquid nitrogen. Thesegases can be frozen in liquid nitrogen, and samples or substances to berapidly frozen can be placed in physical contact with the solidified orslushed gas produced. The solid gas used for initial studies was argon(freezing point=−189.2° C.). Because it is a noble gas, it isnon-reactive and relatively safe to handle.

Solid/slushed gas technology was used to vitrify water and freezebiological specimens. Cryostats were filled with liquid nitrogen andliquid argon from Liquid-Air Corp in Phoenix, Ariz. Liquid argon waspoured into Pyrex test tubes (10-12 cc), which were then lowered into aDewer flask containing liquid nitrogen. The argon sample froze solid in1 to 2 minutes, and the solid cylinder of argon was dumped onto aStyrofoam tray. A silver wire loop 1 cm in diameter was dipped intodistilled water, then rapidly plunged manually into the solid argon. Thewater film shattered into several droplets of instantly frozen ice.Initial biological specimen studies were performed using 2-cell mouseembryos. The same silver wire loops were clipped into culture mediacontaining the embryos, and a liquid film of media containing at leastone embryo was obtained in the loop. This was rapidly and manuallyplunged into solid argon cylinders, achieving hyper-rapid freezing inthe form of small shattered droplets less than 1 mm diameter. A total ofeight mouse embryos were frozen in this manner, and microscopicinspection of the embryos upon thawing revealed that four had zonafracture and cell membrane rupture damage, and the other four wereintact and visually undamaged.

A container for the liquid or biological sample is unnecessary. Smallbare droplets of the sample can be dropped directly into the cryogenicslush or onto the solidified gas. Chemical contamination or damage tothe sample does not occur, especially if a noble gas such as argon isused for the refrigerant. The small shattered film droplets from thefirst experiments demonstrated the feasibility of this innovation, sotechnical designs for dropping small liquid drops of protein orbiological samples onto solid cryogenic gases or slushes were made, withthe limiting factor of acceleration of small drops by gravity throughair. Very small drops arrive more slowly to the refrigerant surface,passing through a slower temperature gradient and increasing thefreezing time (and ice crystal size). In order to overcome thislimitation, a nebulizer was used to produce a fine spray of liquidsample with extremely small droplet sizes, then accelerating the finespray rapidly into the slush or solid refrigerant surface. Vitrificationof water is expected because droplet diameters can be less than thewater film thickness achieved in the examples in the referencepublications, in which vitrification was not achieved. However, thesmaller the droplet the more susceptible it is to the temperaturegradient near the refrigerant surface. If the surface is not approachedrapidly enough, the small droplet will pre-freeze slowly in the airbefore making contact with the surface, resulting in larger ice crystalformation. The advantages of the solid-to-liquid phase change inhyper-rapid freezing applies only to droplets that penetrate the surfaceof a solidified gas or slush in the liquid state. Nebulized samplesshould therefore be sprayed or driven onto the refrigerant surface veryrapidly.

Another concern regarding very small nebulized droplets issusceptibility to rapid evaporation. The increased surface to volumeratio of small droplets allows a rapid phase change from liquid to gas,so the droplet could evaporate entirely before it reaches therefrigerant surface.

The improved designs of the nebulizer freezing method include use of ahigh humidity or water saturated atmosphere, and very fast sprayvelocities to maximize the life-span of the droplets. Rapid sprayvelocities limit the amount of time for evaporation to occur, andincrease the temperature gradient near the refrigerant surface, reducingthe chance of precooling the sample. Evaporation of a liquid droplet isincreased on the forward side when it is sprayed into a stillatmosphere. Therefore, further refinements of the designs involvespraying nebulized droplets into a stream of air moving in the samedirection at about the same velocity, with the air stream and nebulizedstream directed onto the refrigerant surface.

Some fragile protein or biological samples may be damaged or denaturedby the high fluid shear forces of the nebulizer. Low initial nebulizerspray velocities and high air stream velocity can be used to acceleratethe slow droplet to high surface-impact speeds, while protecting thesample from high fluid shear forces.

A typical technical design is a hollow metal sphere filled with liquidargon, then immersed in liquid nitrogen (FIG. 2). Once argon freezesonto the inner rim of the sphere, the container is withdrawn and thecenter core of remaining liquid argon is poured out through a hole inthe top of the container, leaving a shell of solid argon frozen to theinner surface. A nebulizer head is then passed through the hole, to thecenter of the sphere. The liquid sample is sprayed onto the solid argon,rapidly freezing the nebulized droplets into tiny ice spheres. Samplefusion heat melts the solid argon, and the liquid argon then flows onthe bottom of the sphere, collecting into a pool and washing the frozensample droplets to a central location. The frozen sample consists of alarge number of tiny ice particles with undamaged protein, biological,or other substances imbedded inside. These can be recovered, or left andstored in the liquid argon, which is now used as a storage refrigerant.

Other technical designs involve spraying nebulized samples onto an argonslush or onto sheets of frozen argon. A mass production design uses ahollow metal cylinder filled with continuously circulating liquidnitrogen, with the horizontal cylinder half submerged in a pool ofliquid argon (FIG. 3). Slow rotation of the cylinder allows a shell offrozen argon to coat the outside surface, upon which a continuous sprayof liquid nebulized sample is driven. Frozen sample droplets in liquidargon then flow into the pool of liquid argon to be collected.

Hyper-rapid freezing of liquids (or vapors) by nebulized droplet contactwith solidified or slushed cryogenic gases has several features andadvantages. These novel features include:

The solid-to-liquid phase change of the cryogenic “gas” refrigerantallows a high density liquid medium to surround the freezing sampledroplets. A liquid medium has a much higher thermoconductivity than alow density gas medium, so heat is transferred more rapidly andefficiently out of the sample droplet, resulting in a higher freezingrate than samples surrounded by a liquid-to-gas refrigerant phasechange. The cryogenic refrigerant solid-to-liquid phase change rapidlyabsorbs heat and efficiently transfers heat from the liquid or vaporsubstance being frozen. The liquid substance to be frozen makes directcontact with the surface of the refrigerant, typically being completelyor partially surrounded by the liquefied refrigerant, thus increasingthe surface area available for heat flux into the refrigerating solid orslush and out of the freezing sample. Freezing rates are dramaticallyincreased by the extremely high surface to volume ratio of smalldroplets in the nebulized sample, or by the direct application of avaporized sample. Rapid heat flux is further increased by direct contactbetween the refrigerant and the liquid sample. Freezing rates aremaximized without an intervening layer of another substance, such as acontainer or conduit wall.

The solid-to-liquid refrigerant phase change coupled to the highsurface-to-volume ratio of nebulized droplets produces extremely rapidfreezing rates, which allows minimal or no ice crystal formation andminimal osmotic damage to liquid biological samples. The hyper-rapidfreezing method is easily expandable to allow large scale freezing oflarge volumes of liquid samples. Devices designed to use this processcan be readily scaled up for larger sample volumes because of thecontinuous nature of the process, and the adaptability of the process tothe use of multiple parallel basic devices.

The hyper-rapid freezing process can be applied in a continuous mannerand is easily adapted to economies of scale by the use of multipleparallel devices, resulting in low costs for small volumes or largevolumes of frozen samples. The surface of solidified gas refrigerant canbe continuously renewed by systems using rotating drums, extruded plugs,etc., and a slushed gas refrigerant surface can be renewed by continuousflow. These systems produce a constantly-available fresh surface ofrefrigerant to allow continuous freezing of a steady stream of nebulizedsamples. Large scale freezing is further enhanced by the low cost ofsome refrigerant raw materials (e.g. liquid nitrogen and liquid argon).

Nebulized sample particles frozen using this method are easily recoveredbecause they remain suspended in the liquefied primary refrigerant atthe end of the freezing process. The primary refrigerant liquefies afterabsorbing heat from the sample, then washes the frozen sample dropletsaway from the active freezing contact site. Depending on the differencein density between the frozen sample particles and the liquefiedrefrigerant, the sample particles can be recovered 1) by gravity at thebottom of the refrigerant vessel, 2) by skimming or overflow afterfloating to the refrigerant surface, or 3) by filtration if suspendedwithin the refrigerant. The ease of sample recovery is a distinctadvantage over other prior-art methods, which require scraping frozensamples off cold metal surfaces, removing samples from porous or thinfilm plates, detaching samples from metal grids, or unsealing samplesfrom tubes or metal canisters. Short or long-term storage of frozennebulized samples is also greatly simplified by this freezing method,again because the sample remains within the liquefied primaryrefrigerant after freezing. As outlined above, the primary refrigerantis generally nonreactive and nontoxic, typically liquid nitrogen or aliquefied noble gas, so the frozen sample is subjected to no long-termadverse effects if it remains suspended in the primary refrigerant forstorage. An additional advantage is that the sample remains at itsfreezing temperature indefinitely because there is no need to transferto another container or medium for storage, so the risk of heating thesample above one of its glass or crystalline transition temperatures isminimized.

Solid or slushed cryogenic “gas” refrigerants used in this method may benoble gases (He, Ne, Ar, etc.) or common industrial cryogens (nitrogen)which are generally chemically nonreactive and nontoxic. This isespecially useful when freezing organic or biological materials, orliving cells, because the freezing samples come into direct contact withthe refrigerants, and some of the refrigerant substance is expected todiffuse into the frozen samples. The use of nonreactive and nontoxicrefrigerants is a significant advantage over methods that use organicsolvents such as ethane, propane, and butane as liquid refrigerants.Frozen samples are heavily contaminated by these organic solvents, whichpoison the sample or require removal (usually by a less-toxic organicsolvent). Use of noble gases, or in some instances nitrogen, isespecially useful for “time stopping” experiments using samples ofactively reacting chemicals.

Primary (solid-to-liquid phase) refrigerants and secondary refrigerants(those used to initially freeze the primary refrigerants) are typicallyinexpensive atmospheric or industrial cryogenic liquefied gasescurrently mass-produced by efficient industries. They include liquidnitrogen, liquid argon, and liquid helium, and can be extended to moreexotic refrigerants such as liquid hydrogen, liquid neon, liquid oxygen,alcohol, or even chilled metals such as mercury. Most of therefrigerants are abundant, easy to ship and handle, and are inert,reducing the cost of purchasing or using the primary raw materials ofthe process. Waste products generated by the process, typicallyatmospheric gases or helium, are simply vented or recycled, or forhydrogen, simply burned to produce water vapor, so disposal costs ofwaste products are minimal or absent. The low cost of operation and thewide availability of raw materials makes this process less expensivethan other hyper-rapid freezing methods. Most nebulizers are inexpensivepressure spray nozzles or common ultrasonic commercial devices. Theserelatively inexpensive items represent a significant cost advantage overcurrently used rapid freezing devices containing high pressure vessels,programmable electronic variable rate cooling systems, mechanical orelectromagnetic sample plungers, or elaborate chemical preparation ofsamples. Another cost advantage of this freezing process is the absenceof organic solvent or waste disposal—the refrigerants are simplyrecycled or are vented off into the atmosphere.

This freezing process has a minimal number of working components: 1. asample storage and delivery system, 2. nebulizer or atomizer, 3. primaryfrozen or slushed refrigerant, 4. secondary refrigerant, 5. samplerecovery system, and 6. appropriate insulation. The number of movingparts is also minimized, with the simplest configuration consisting of amoving stream of nebulized sample droplets directed against a stationaryprimary refrigerant. The process simply requires that a high velocitystream of liquid be directed onto a surface of solid or slushedcryogenic gas. More complicated procedures used in currently availablefreezing methods, such as programmable freezing curves, elaborate samplepreparations, or ice crystal seeding steps, are avoided. Because theprocess is simple, equipment costs are reduced, along with equipmentfailure rates and maintenance requirements. More complete automation ispermitted by the simplicity of the process, and the high speed inherentin freezing single samples allows rapid production and delivery comparedto other currently used methods.

Basic control of this freezing process is easily accomplished by simpleadjustments of a few inherent variables. The rate of freezing isdetermined by the temperature gradient applied to the nebulized droplet,and this is easily controlled by changing the velocity of the nebulizedspray onto the primary refrigerant, the temperature of the primaryrefrigerant, or the composition of the primary refrigerant and itsassociated properties of melting point, thermoconductivity, and purity.Spray impact velocity can also be easily controlled by changing thedistance between the nebulizer and the primary refrigerant, as thenebulized stream will usually lose velocity with increasing distancefrom the nebulizer. A notable subtype of this control is using anebulizer-refrigerant distance of zero, i.e. spraying the nebulizeddroplets directly into the slushed or solid primary refrigerant, whichwould have the additional benefit of increasing chaotic flow and heattransfer in the nebulized stream. Control of freezing rate and heattransfer is also easily achieved by changing the size of the nebulizeddroplets, and thus, their surface-to-volume ratio, by using differenttypes of nebulizers or atomizers, changing nebulizer aperture size, oraltering the nebulizer fluid pressure. Addition of cryopreservativesolutions to sample fluids is an optional method to reduce ice crystalsize or formation. Various cryopreservatives may be used, includingdimethyl sulfoxide (DMSO), dextran, sucrose, 1,2 propanediol, glycerol,sorbitol, fructose, trehalose, raffinose, hydroxyethyl starch, propyleneglycol, 2-3 butane diol, polyvinylpyrrolidone (PVP), proline, humanserum albumin, and combinations thereof. Easy control of the freezingparameters using this method represents a distinct advantage over otherprior-art methods.

Different rates of freezing are required to obtain the desired outcomefor various liquid samples, depending on the solvent, concentration,requirement for vitrification, requirement for size of ice crystals, ornebulization limitations. The freezing rate generated by the process canbe easily adjusted to satisfy these requirements by selecting the properprimary refrigerant (e.g. solid nitrogen vs. solid argon) or secondaryrefrigerant (e.g. liquid helium to freeze argon or alcohol), or bychanging the size of the nebulized sample droplets. Other adjustablevariables that change freezing rates include the velocity of thenebulized stream, and the use of a solid vs. slushed refrigerant. All ofthese adjustments can be designed into a single freezing device, whichcould then be used to freeze a wide variety of substances and sampleswith different freezing rates.

The process allows easy interchange or replacement of its basiccomponents—primary and secondary refrigerants and nebulizers. Drainageand replenishment of refrigerants is accomplished by pumps or gravityfill, and waste gases are removed by vents. Nebulizers, atomizers, flowcytometers and vapor generators can be easily changed to produce thedesired spray volume, rate, or droplet size.

EXAMPLE 1

A rapid stream of nebulized water droplets was achieved. The dropletswere hyper-rapidly frozen by directing the stream onto the surface offrozen argon. The frozen droplets were collected in the liquid primaryrefrigerant.

Equipment

1 Styrofoam ice chest (1 gallon)

nebulizer—ultrasonic

box fan (5 inch) 120 volt 60 Hz

1 Styrofoam platter with concave hemispheric surface

Pyrex glass test tubes—red-toe large 20 ml

open mouth Dewer flask

1 vacuum thermos bottle

1 foam thermos cup (250 ml)

liquid nitrogen

liquid argon

A ½ inch diameter hole was drilled through the ice chest wall ½ way upthe side to form a side port. A 4½ inch square was cut out of one end ofthe Styrofoam ice chest lid and 4 holes (½ inch) were drilled into thelid at the opposite end. The box fan was embedded in the square hole andtaped in place, and the fan lead wires were soldered to a transectedelectrical extension cord. A tape strap was placed over the 4 holes inthe lid, with control of forced air achieved by removal of tape from 0,1, 2, 3, or all 4 holes. The ultrasonic nebulizer was placed under 4inches of water inside the Styrofoam ice chest.

The fan and ultrasonic nebulizer were turned on, achieving awell-controlled very rapid stream of nebulized mist through the sideport of the ice chest. The mist stream direction was easily controlledby placing a glass pipette through the side port.

Six Pyrex glass test tubes were precooled in liquid nitrogen, then ¾filled with liquid argon (approximately 12 cc-15 cc). The test tubeswere partially immersed in liquid nitrogen for 10 minutes, and 90% to100% of the argon was frozen solid in each tube. The Styrofoam workingtray contained an approximate 8 inch hemispheric depression. Thedepression was precooled with liquid nitrogen for 2 minutes. All liquidnitrogen was then poured out of the working tray, and the six test tubesof frozen argon were then emptied into the working tray depression.Solid frozen argon was in the form of transparent cylinders ofapproximately 12 cc initial volume each.

The Styrofoam working tray was tilted approximately 30°, allowing thefrozen argon cylinders to remain in the middle of the depression, andmelted argon flowed off center to a collection area at the lowest point.The stream of nebulized mist from the side port of the ice chest wasthen directed against the frozen argon cylinders using the glass pipettefor stream control. The movement of the nebulized mist stream was rapid(estimated 1 to 1½ meters/sec), and distance of stream pipette to frozenargon surface was 10 cm.

Nebulized water droplets froze on impact with solid argon into tiny iceparticles. The frozen argon cylinders melted entirely in approximately40 seconds, when exposed to the mist stream, and liquid argon flowed tothe collection area at the bottom of the working tray. Ice particleswere collected as a “scum” floating on the surface of the liquid argon.The liquid argon evaporated after 5 minutes.

Example 1 Conclusions

1. A rapid stream of nebulized water droplets (estimated 1 to 1½ m/sec)can be generated by forcing air into a small ultrasonic nebulizationchamber using a fan, with a ½ inch exit port placed on the opposite endof the chamber from the fan.

2. The speed and direction of the nebulized water stream can becontrolled by changing the internal pressure of the nebulizationchamber, and by placing an adjustable pipette in the exit port.

3. Argon frozen into 12 cylinders melted completely in approximately 40seconds when exposed to a stream of nebulized water particles in air at74° F. (when lying on a precooled Styrofoam surface).

4. Nebulized water droplets are rapidly frozen into tiny ice particlesupon contact with solid argon, when driven as a “mist stream” onto afrozen argon surface at 1 to 1½ m/sec.

5. The tiny ice particles float on the surface of liquid argon in theform of a “scum” of coalescing particles, and can be collected bypooling the melted argon at a lower level than the frozen argoncylinders.

EXAMPLE 2

The heat of sublimation of a solid primary refrigerant (carbon dioxide)was used to rapidly freeze nebulized water droplets. Water droplets werefrozen by directing a rapid nebulized stream against the surface of dryice (frozen carbon dioxide).

Equipment

1 Styrofoam ice chest (1 gallon)

nebulizer—ultrasonic

box fan (5 inch) 120 volt 60 Hz

1 Styrofoam platter

2 lbs dry ice

A similar method as that described in Experiment 1 was used to producean air driven stream of nebulized water mist (estimated 1 to 1½ m/sec)through the side port glass pipette. The differences were as follows. Adry ice block was placed on the Styrofoam platter. The stream ofnebulized mist was directed against the top surface of the dry ice blockat a distance of approximately 10 cm for 60 seconds. During this time amoderate amount of condensed “steam” was blown radially away from thenebulized stream contact point on the dry ice surface. Presumably this“steam” was condensed water vapor from around the nebulized droplets inthe stream. Frozen ice droplets were not observed bouncing away from thecarbon dioxide surface. Nebulized water droplets froze on impact withthe surface of the dry ice into tiny ice particles. These particlescollected together as a white flaking “scum” along the edges of theshallow crater formed in the dry ice block at the contact point with thenebulized stream.

Example 2 Conclusions

1. When driven as a “mist stream” onto a frozen carbon dioxide surface,nebulized water droplets are rapidly frozen into tiny ice particles.

2. The heat absorbed by carbon dioxide with the solid-to-gas phasechange can be used to rapidly freeze small water droplets.

3. The tiny ice particles collect into a white flaking “scum” on thesurface of the dry ice around the nebulized stream contact point.

Successful hyper-rapid freezing of nebulized water droplets was achievedusing the solid-to-gas phase change heat release of sublimation.

EXAMPLE 3

The solid-to-liquid phase of a cryogenic gas may be used to absorb heatfrom an oocyte in cell culture media rapidly enough to vitrify the cell.An oocyte is placed in a small biological storage vial containing cellculture media, then plunged directly into a vat of solid/liquid nitrogenslush. Heat is very rapidly absorbed out of the vial (and oocyte) andinto the slushed nitrogen without an intervening insulating gas envelopegenerated around the vial. The heat would be consumed as heat of fusionin the solid to liquid nitrogen phase change, so a nitrogen gas layerwould not form around the vial and slow the conduction of heat out ofthe vial. The high thermoconductivity of the process should besufficient to increase the freezing rate to allow vitrification of theculture media and the oocyte. Modifications of the system, such as usinga copper or silver vial, could be applied to maximize thermoconductivityand freezing rate. Without ice crystal or hyper-concentrated solutedamage, the oocyte should survive intact and fertilize normally.

Although the preferred embodiment of the apparatus of the invention hasbeen described above in some detail, it should be appreciated that avariety of embodiments will be readily apparent to one skilled in theart. The description of the apparatus of this invention is not intendedto be limiting to this invention, but is merely illustrative of thepreferred embodiment.

While the methods for sample preservation and storage have beendescribed in terms of preferred embodiments, it will be apparent tothose skilled in the art that variations and modifications may beapplied to the methods and in the steps or sequence of steps of themethods described herein without departing from the concept, spirit andscope of the invention.

I claim:
 1. A method for freezing a liquid comprising transforming saidliquid into very small drops, rapidly driving said very small dropsdirectly onto a refrigerant which is at least partially solidified, andcollecting the frozen liquid.
 2. The method of claim 1 wherein said atleast partially solidified state of said refrigerant is achieved byevaporative cooling in a vacuum chamber.
 3. The method of claim 1wherein said refrigerant is solid.
 4. The method of claim 3 wherein saidsolid refrigerant is undergoing sublimation.
 5. The method of claim 1wherein said transformation into very small drops is accomplished by anebulizer.
 6. The method of claim 1 wherein said transformation intovery small drops is accomplished by a flow cytometer.
 7. The method ofclaim 1 wherein said refrigerant is a slush comprising a mixture ofsolid and liquid.
 8. The method of claim 7 wherein said solid and liquidare the same element or compound.
 9. The method of claim 7 wherein saidsolid and liquid are different elements or compounds.
 10. A method forpreserving a sample suspension of biological material comprisingtransforming said sample suspension into very small drops, rapidlydriving said very small drops directly onto a refrigerant which is atleast partially solidified, and collecting the frozen sample.
 11. Themethod of claim 10 wherein transforming the sample suspension into verysmall drops is accomplished by a nebulizer.
 12. The method of claim 10wherein transforming the sample suspension into very small drops isaccomplished by a flow cytometer.
 13. The method of claim 10 whereinsaid refrigerant is a solid.
 14. The method of claim 10 wherein saidrefrigerant is a slush comprising a mixture of solid and liquid.
 15. Themethod of claim 14 wherein said solid and liquid are the same element orcompound.
 16. The method of claim 14 wherein said solid and liquid aredifferent elements or compounds.
 17. The method of claim 10 wherein therefrigerant is inert and nonreactive.
 18. The method of claim 17,wherein the inert and nonreactive refrigerant is a noble gas.
 19. Themethod of claim 18, wherein the noble gas is argon.
 20. The method ofclaim 17, wherein said inert and nonreactive refrigerant is nitrogen.21. The method of claim 20 wherein said nitrogen is a slush and isachieved by placing liquid nitrogen under a partial vacuum.
 22. Themethod of claim 10 wherein the refrigerant is solid carbon dioxide. 23.The method of claim 10 wherein the refrigerant comprises a primary andsecondary refrigerant, wherein the secondary refrigerant is held at atemperature below the freezing point of the primary refrigerant, saidsecondary refrigerant achieving the solid or slushed state of theprimary refrigerant through thermal contact with the primaryrefrigerant.
 24. The method of claim 23 wherein said primary refrigerantis argon and said secondary refrigerant is nitrogen.
 25. The method ofclaim 10 wherein a cryopreservative is added to the sample prior to thestep of driving the sample onto refrigerant.
 26. The method of clam 10wherein said refrigerant is oxygen or hydrogen.
 27. The method of claim10 wherein said refrigerant is organic.
 28. The method of claim 27wherein said organic refrigerant is selected from the group consistingof liquid carbon dioxide, propane, butene, ethane, Freon, and ethanol.29. The method of claim 10 wherein said refrigerant is mercury or lead.30. The method of claim 10 wherein said refrigerant is selected from thegroup consisting of helium, neon, nitrogen, argon, krypton, and xenon.31. A method of stopping a liquid chemical or biological reactioncomprising rapidly cooling the reaction mixture such that the chemicalor biological activity stops; wherein said cooling is achieved bytransforming the reaction mixture into very small drops, driving thevery small drops directly onto a refrigerant which is at least partiallysolidified, and collecting the cooled reaction mixture.
 32. The methodof claim 31 wherein a nebulizer is used to achieve the very small dropsof liquid reaction mixture.
 33. The method of claim 31 wherein a flowcytometer is used to achieve the very small drops of liquid reactionmixture.
 34. A method for lyophilizing a suspension of compounds orcompositions while retaining structural integrity comprising: (a)rapidly driving the suspension directly onto a refrigerant to freeze thesuspension, wherein said refrigerant is at least partially solidified;(b) collecting the frozen suspension; and (c) drying the frozensuspension.
 35. The method of claim 34 further comprising the step oftransforming the suspension into very small drops prior to the step ofdriving the suspension onto the refrigerant.
 36. An apparatus forfreezing a sample suspension, including biological samples, comprising ameans for rapidly driving the sample suspension through an outlet, and acontainer for refrigerant positioned such that the outlet delivers thesample directly onto the refrigerant and the refrigerant is at leastpartially solidified.
 37. The apparatus of claim 36 further comprising ameans for transforming the sample suspension into very small drops. 38.The apparatus of claim 36 wherein said refrigerant is solid.
 39. Theapparatus of claim 36 wherein said refrigerant is a slush comprising amixture of solid and liquid.
 40. An apparatus for freezing a suspensionof biological material comprising a flow cytometer positioned over acontainer holding refrigerant, wherein when activated, the flowcytometer transports the suspension of biological material directly ontothe refrigerant.
 41. The apparatus of claim 40 wherein said refrigerantis solid.
 42. The apparatus of claim 40 wherein said refrigerant is aslush comprising a mixture of solid and liquid.
 43. The apparatus ofclaim 40 further comprising a container containing the suspension ofbiological material, said container communicatively coupled to the flowcytometer.
 44. An apparatus for freezing a suspension of biologicalmaterial comprising a nebulizer and a container holding refrigerant,wherein when activated, the nebulizer transports the suspension ofbiological material directly onto the refrigerant, wherein saidrefrigerant is at least partially solidified.
 45. The apparatus of claim44 further comprising a container containing the suspension ofbiological material, said container communicatively coupled to thenebulizer.
 46. The apparatus of claim 44 wherein said container holdingrefrigerant comprises a container holding a primary and a secondaryrefrigerant, wherein the secondary refrigerant is held at a temperaturebelow the freezing point of the primary refrigerant, said secondaryrefrigerant achieving at least a partially solidified state of theprimary refrigerant through thermal contact; wherein the nebulizertransports the suspension of biological material directly onto theprimary refrigerant.
 47. The apparatus of claim 46 comprising separatecontainers for the primary and secondary refrigerant, wherein saidcontainer of secondary refrigerant is in thermal contact with theprimary refrigerant, said secondary refrigerant causing said primaryrefrigerant to achieve at least a partially solidified state.
 48. Anapparatus for preserving a sample suspension comprising: (a) a samplechamber communicatively coupled to a nebulizer; (b) a container holdingrefrigerant which is at least partially solidified; and (c) a sampleoutlet; wherein said nebulizer, when activated, transports the liquidsample from the sample chamber through the sample outlet directly ontothe refrigerant.
 49. The apparatus of claim 48 wherein the samplechamber has openings for regulating the speed of the sample exiting thesample chamber through the sample outlet.
 50. The apparatus of claim 48wherein the sample chamber is associated with a fan for regulating thespeed of the sample exiting the sample chamber through the sampleoutlet.
 51. The apparatus of claim 48 wherein said at least partiallysolidified refrigerant is achieved through a primary refrigerant in acontainer which is in thermal contact with a secondary refrigerant heldat a temperature below the freezing point of the primary refrigerant,causing the primary refrigerant to achieve at least a partiallysolidified state, wherein the nebulized sample directly contacts theprimary refrigerant.
 52. An apparatus for freezing a sample suspension,including biological samples, comprising a means for transforming thesample suspension into very small drops, means for creating a saturatedor humidified gas medium flowing onto refrigerant which is at leastpartially solidified, means for rapidly driving the very small drops ofsample suspension; within the gas medium, through an outlet, and acontainer for the refrigerant, wherein the sample outlet delivers thesample directly onto the refrigerant.
 53. An apparatus for freezing asample suspension, including biological samples, comprising a means fortransforming the sample suspension into very small drops, means forrapidly driving the very small drops of sample suspension, through anoutlet, and a container for the refrigerant, wherein the sample outletdelivers the sample directly onto the refrigerant, wherein said meansfor rapidly driving the very small drops of sample suspension through anoutlet comprises a nebulizer attached to a sample delivery means, andwherein said container for refrigerant comprises a hollow sphere forholding primary refrigerant, with an opening for inserting saidnebulizer, said sphere positioned within a container for secondaryrefrigerant such that the sphere is at least partially submerged in thesecondary refrigerant.
 54. An apparatus for freezing a liquid sample,including biological samples, comprising: (a) a rotating drum filledwith liquid secondary refrigerant, said drum positioned inside a housinghalf-filled with liquid primary refrigerant such that said drum ispartially submerged in the primary refrigerant; and (b) one or morenebulizers positioned over the exposed surface of the drum; wherein inuse, as the drum rotates, the secondary refrigerant inside causes alayer of frozen primary refrigerant to form on the exposed surface, andsample is sprayed from said nebulizers onto frozen primary refrigeranton the surface of the drum, as the drum rotates, the frozen samplefloats off into the liquid primary refrigerant.
 55. An apparatus forfreezing a liquid sample, including biological samples, comprising: (a)a housing with a sloped upper surface comprising grooves, the end wallsof the grooves having openings connected to a pipe at the upper end, andto a trough at the lower end, said housing being hollow with inlet andoutlet ports for circulating liquid secondary refrigerant through thehousing; and (b) multiple nebulizers, one positioned over each groove;wherein liquid primary refrigerant is transported from the pipe throughthe openings, down the grooves where it freezes due to the liquidsecondary refrigerant circulating through the housing, and sample issprayed onto the frozen primary refrigerant in the grooves, causing therefrigerant to melt and flow into the trough.
 56. An apparatus forfreezing a liquid sample, including biological samples, comprising: (a)a housing, said housing divided into first and second sections, thefirst section filled with liquid secondary refrigerant, and the secondsection partially filled with liquid primary refrigerant; (b) a rotatingmetal disk positioned half in the first section and half in the secondsection such that the disk is in contact with both the primary andsecondary refrigerants, said secondary refrigerant cooling the metaldisk causing a layer of frozen primary refrigerant to form on theexposed surface of the disk; and (c) one or more nebulizers positionedin the second housing section containing primary refrigerant such thatsample is sprayed from the nebulizers onto the frozen primaryrefrigerant coating the exposed portion of the disk.
 57. An apparatusfor freezing a liquid sample, including biological samples, comprising:(a) a vertical cylindrical pipe within a larger cylindrical container,said container comprising a lower section filled with liquid secondaryrefrigerant and an upper section, said pipe positioned such that a topopening of the pipe is in the upper section of the cylindrical containerand a bottom opening of the pipe is below the lower section of thecylindrical container; (b) insulation surrounding the cylindricalcontainer; (c) a conduit connected to the lower section of thecylindrical container, said conduit transporting the liquid secondaryrefrigerant; and (d) a nebulizer positioned over the top opening in thepipe; wherein liquid primary refrigerant is forced under pressure upinto the pipe where it freezes as it passes through the lower section ofthe cylindrical container filled with secondary refrigerant, and saidfrozen primary refrigerant is extruded out of the top opening of thepipe, said sample is sprayed onto the extruded primary refrigerant. 58.An apparatus for freezing a liquid sample, including biological samples,comprising a centrifuge with paddles holding solid primary refrigerantconnected to the rotor, and one or more nebulizers positioned in thewalls of the centrifuge such that sample delivered to the nebulizers issprayed onto the solid refrigerant as the rotor turns.
 59. An apparatusfor freezing a liquid sample, including biological samples, comprising acolumn attached to a rotating head comprising multiple horizontalnebulizers positioned over a cylindrical container, the walls of whichare coated with solid refrigerant; sample is delivered to the nebulizersthrough the column and sprayed onto the solid refrigerant coating thewalls of the container as the head rotates.
 60. An apparatus forfreezing a liquid sample, including biological samples, comprising: (a)a housing filled with liquid secondary refrigerant; (b) one or morehollow rotating cylinders positioned such that they are submerged in theliquid secondary refrigerant; (c) inlet tubes for sample transferconnected to respective cylinders; (d) nebulizers positioned on saidinlet tubes inside the cylinders to deliver sample to the inside wallsof the cylinders; wherein liquid primary refrigerant is placed insidethe cylinders, and as the cylinders rotate, the primary refrigerantfreezes on the walls; sample sprayed onto the cylinder wall freezes anddrops into the remaining liquid refrigerant at the bottom of therotating cylinder.
 61. An apparatus for freezing a liquid sample,including biological samples, comprising a rotating cylindricalreceptacle with solid refrigerant coating the inner walls, and a sampledelivery tube with an injection aperture at the end; wherein saiddelivery tube is positioned such that the injection aperture is in closeproximity to the solid refrigerant coating the receptacle walls.
 62. Anapparatus for freezing a liquid sample, including biological samples,comprising: (a) a receptacle holding slushed refrigerant; (b) a cylinderwith small injection apertures located at one end, positioned in thereceptacle such that the injection apertures are submerged in theslushed refrigerant; wherein pressurized sample is delivered through theinjection apertures in the cylinder into the slushed refrigerant.
 63. Anapparatus for freezing a liquid sample, including biological samples,comprising: (a) a receptacle holding slushed refrigerant, saidreceptacle having small injection apertures in the bottom; and (b) aninjection apparatus connected to the bottom of said receptacle, saidinjection apparatus comprising nebulizer jets; wherein sample isinjected into the slushed refrigerant in the receptacle by means of saidnebulizer jets.
 64. An apparatus for freezing a liquid sample, includingbiological samples, comprising a container for refrigerant positionedinside a vacuum chamber, said vacuum chamber having a nebulizerconnected thereto, wherein the nebulizer is positioned over thecontainer for refrigerant.